HumaTex CRP. Design Verification. Contents

Transcription

1 Design Verification HumaTex CRP Contents 1 Function Reproducibility Sensitivity and dynamic range... 2 Preparation of serum control panel... 2 Sensitivity test results... 2 Prozone check... 3 Function test with kit controls... 3 Specificity check with in-house panel sera Interferences... 3 Results Specificity and sensitivity... 4 Results Stability... 4 Real time stability... 4 Results... 5 Format: Rev. 010 valid of /5

2 1 Function The HumaTex CRP latex agglutination slide test has been designed as a qualitative and semi-quantitative test for the rapid determination of C-reactive protein (CRP) in native undiluted serum samples. The test is based on latex agglutination technique, featuring polystyrene latex which has been coated with monospecific antibodies (goat) against human CRP. If CRP positive sera react with the latex suspension, visible agglutinates are formed within 2 minutes. Positive and negative controls are incorporated in the kit to perform function checks and to compare the results obtained with unknown samples. For semi-quantitative determinations the serum samples are diluted stepwise with glycine saline buffer. The last dilution step which give a positive result (agglutination) is used to calculate the CRP concentration in the sample. 2 Reproducibility The within-run reproducibility of the HumaTex CRP (lot H2133, exp date ) latex agglutination slide test was calculated from 10-fold determinations of positive and negative samples. The day-to-day reproducibility was calculated from the results obtained on 4 different dates (HumaTex CRP, lot H2143, exp date ). 10 positive and 10 negative control sera were employed as sample materials. HumaTex CRP showed in all cases a very good reproducibility. 3 Sensitivity and dynamic range Description of control materials In-house standard is calibrated against a standard material (ICC Multi-Level CRP Calibrator). Standard dilutions are prepared by diluting the in-house standard with glycine buffered saline. For prozone phenomenon check a high concentrated in-house standard material is employed (about 800 mg/l CRP). The following standard dilutions are prepared: No CRP, mg/l The kit controls positive and negative are employed for function test. The positive control is typically adjusted to a CRP concentration of mg/l. Preparation of serum control panel A panel of each 10 sera, CRP positive and negative, has been established and confirmed with an independent test method. Each individual panel material has been aliquoted and kept deep-frozen. Before use, the appropriate number of panel sera are removed from the freezer and brought to room temperature. Once thawed, panel sera must not be frozen again but have to be discarded after use. Sensitivity test results All concentrations are tested in double. The difference in agglutination degree must not exceed one titer step. No. CRP, mg/l Agglutination degree at 2 min / /- / / Intensity grades: 4+ (very strong agglutination); 3+ (strong agglutination); 2+ (medium agglutination); 1+ (visible); (borderline); - (no agglutination) Rev /5

3 Prozone check For prozone check the high concentrated in-house standard preparation is employed in the following concentrations: No. CRP, mg/l Agglutination degree at 2 min / /2+ Although prozone phenomenon can be recognized at concentrations above 500 mg/l, up to 1000 mg/l CRP positive agglutination results will be obtained. Function test with kit controls The kit controls are employed for a function test of the latex reagent. Positive control Negative control Agglutination degree at 2 minutes 4+ - Serial dilutions of the positive control typically show the following agglutination degrees: Controls Expected agglutination degree at 2 minutes Undiluted positive control 3+ 1/2 diluted positive control > ± 1/4 diluted positive control 1/8 diluted positive control - The titer for the positive kit control is usually 1:4. Specificity check with in-house panel sera Predetermined in-house panel sera have been employed for a specificity check. 4 Interferences No. Positive panel Negative panel Description of materials Interferences have been tested for bilirubin, lipid, hemoglobin and ascorbic acid prepared in a human serum pool tested negative for CRP and RF. These serum preparations were spiked with CRP resulting in a borderline agglutination (+/-). Rev /5

4 Results Substance Serum pool w/o CRP Serum pool with CRP Serum pool (control) neg +/- to 1+ Bilirubin (40 mg/dl) neg +/- Lipids ( 1000 mg/dl) neg +/- Hemoglobin (500 mg/dl) neg +/- Ascorbic acid (500 mg/dl) neg +/- None of the above substances showed any influence on the expected results. 5 Specificity and sensitivity Description of method and specimen The specificity and sensitivity of the HumaTex CRP test has been evaluated by method comparison against a turbidimetric and a latex agglutination test. 88 respectively 75 native sera have been employed in the studies with the turbidimetric and the latex test. Results A) Turbidimetric test method The turbidimetric test showed 31 sera with CPR concentrations between 8 and 95 mg/l CRP. All of these 31 sera were correctly found positive with the HumaTex CRP test. 57 sera showed concentrations below 4 mg/l CRP with the turbidimetric test. All 57 sera were correctly determined as negative with the HumaTex CRP test. BioSystems CRP Test Positive Negative HumaTex CRP Positive 31 0 Negative 0 57 Total From the above a diagnostic specificity for the HumaTex CRP test resulted to 100%. Equally the diagnostic sensitivity was found to be 100%. B) Latex test method The 27 positive sera were found positive with both HumaTex CRP and the competitor method. 45 of 48 negative sera were found negative with HumaTex CRP. 3 sera produced borderline agglutination (1+). Behring CRP Latex Test Positive Negative HumaTex CRP Positive 27 3 Negative 0 45 Total From the above a diagnostic specificity for the HumaTex CRP test resulted to 94%. The diagnostic sensitivity could be calculated to 100%. 6 Stability Real time stability The stability of the HumaTex CRP latex agglutination slide test has been demonstrated on real-time stability studies and is checked by temperature stress studies on each produced lot. Rev /5

5 A function tests on the finalised kit has been performed using standard materials and kit controls. Results of the function tests are summarised below. Latex rgt. lot.: 038 Manuf. date: Aug Result fresh Result on Nov/ / Latex rgt. lot.: 039 Manuf. date: Okt Result fresh Result on Nov/ / Latex rgt. lot.: 040 Manuf. date: Jan Result fresh / Result on Nov/ Latex rgt. lot.: 0130 Manuf. date: May 2003 Result fresh / Result on May / Latex rgt. lot.: 0131 Manuf. date: Jul 2003 Result fresh 3/4+ 3/ / Result on Jun / Latex rgt. lot.: 0136 Manuf. date:aug 2003 Result fresh 4/3+ 3/ / Result on Sep / / Nine different production batches of HumaTex CRP have been checked after different months real time storage (storage conditions 2 8 C). The results are summarised below. LOT Months Storage Standard, mg/l CRP Results Three individual production batches of HumaTex CRP have been checked after 26, 24 and 22 months real-time storage (storage conditions: C). In addition nine different batches have been checked between 6 and 15 months of real time storage (storage conditions 2 8 C). The above results clearly demonstrate the stability of HumaTex CRP and support the stability claim of 20 months. Rev /5